Abstract:

Clustered regularly interspaced short palindromic repeats
(CRISPRs) are composed of an array of short DNA repeat sequences separated by unique spacer sequences that are
flanked by associated (Cas) genes. CRISPR-Cas systems are
found in the genomes of several microbes and can act as an
adaptive immune mechanism against invading foreign nucleic
acids, such as phage genomes. Here, we studied the CRISPRCas systems in plant-pathogenic bacteria of the Ralstonia sola-
nacearum species complex (RSSC). A CRISPR-Cas system was
found in 31% of RSSC genomes present in public databases.
Specifically, CRISPR-Cas types I-E and II-C were found, with
I-E being the most common. The presence of the same CRISPRCas types in distinct Ralstonia phylotypes and species suggests the acquisition of the system by a common ancestor
before Ralstonia species segregation. In addition, a Cas1 phylogeny (I-E type) showed a perfect geographical segregation
of phylotypes, supporting an ancient acquisition. Ralstonia
solanacearum strains CFBP2957 and K60 T were challenged
with a virulent phage, and the CRISPR arrays of bacteriophage insensitive mutants (BIMs) were analysed. No new spacer acquisition was detected in the analysed BIMs. The functionality
of the CRISPR-Cas interference step was also tested in R. solanacearum CFBP2957 using a spacer-protospacer adjacent
motif (PAM) delivery system, and no resistance was observed
against phage phiAP1. Our results show that the CRISPR-Cas
system in R. solanacearum CFBP2957 is not its primary antiviral strategy.